The Detection of Glycosaminoglycans in Pancreatic Islets and Lymphoid Tissues
In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage, and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP–link protein complex is applied to tissue sections and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates.
To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP. The hyaluronan–HABP complexes are extracted and the hyaluronan concentration in the tissue is determined using an ELISA-like assay.
Heparan sulfate is identified in mouse tissues by Alcian blue histochemistry and indirect immunohistochemistry. In human tissues, heparan sulfate is best detected by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A biotinylated secondary antibody is then applied in conjunction with streptavidin-peroxidase and its binding to the anti-heparan sulfate antibody is visualized by enzymatic precipitation of chromogenic substrates.
Key wordsHyaluronan Heparan sulfate Pancreatic islets Lymphoid tissue Hyaluronan binding protein Immunohistochemistry
This research was performed with the support of the Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative type 1 diabetes research project sponsored by JDRF, Grant 25-2010-648, National Institutes of Health grants U01 AI101984, CSGADP Innovative Project (under AI101984), and P01 HL098067 (T.N.W.). Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at www.jdrfnpod.org/our-partners.php. This work was also supported by a National Health and Medical Research Council of Australia (NH&MRC)/Juvenile Diabetes Research Foundation (JDRF) Special Program Grant in Type 1 Diabetes (#418138; C.S.), a NHMRC Project Grant (#1043284), JDRF nPOD Research Grant 25-2010-716 (C.S.), a research grant from the Roche Organ Transplantation Research Foundation (ROTRF)/JDRF (#477554991; C.S.), and a Deutsche Forschungsgemeinschaft (DFG) Research Grant NA 965/2-1 (N.N.). We thank Anne Prins for assistance with the Alcian blue histochemical methodology and Lora Jensen and Sarah Popp for optimizing the heparan sulfate immunohistochemistry on nPOD human pancreas sections.
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